Enabling widespread use of high resolution imaging of oxygen in the brain As. essment of brain oxygenation on the microscopic level has the potential to transform our understanding of important clinical problems, such as stroke, Alzheimer's disease, dementia, chronic hypertension, and brain cancer, facilitating the development of new therapies and helping to improve clinical imaging and treatment protocols. Until now, no technology has been capable of microscopic oxygen imaging in the brain with high spatial and temporal resolution. Over the past several years we have developed a method, termed two-photon phosphorescence lifetime microscopy of oxygen (2PLM), which has the unique capability of fulfilling this niche. This is the only imaging method that allows high resolution mapping of brain oxygenation in real time. We propose to establish a sustainable resource for the dissemination of 2PLM of oxygen, making it available to interested researchers across different fields of neurology and neuroscience. 2PLM is a combination of state-of-the-art two-photon enhanced phosphorescent probes and a unique variant of two- photon laser scanning microscopy. In this project we will optimize and scale up synthesis of the oxygen probes, making them available for interested users. We will incorporate recently developed, dedicated commercial electronic components into 2PLM setups. We will also optimize algorithms and produce user- friendly software for those interested in setting up their own 2PLM systems. In addition, we will organize a resource center where users will be able to receive training and run pilot experiments. The work will be performed at two closely collaborating sites: the University of Pennsylvania (probe chemistry) and MGH (imaging setup). Our laboratories have a long history of productive collaboration. We also collaborate broadly with many researchers across the field interested in oxygen imaging. This will allow us to establish an effective infrastructure for the resource center, making the 2PLM of oxygen technology accessible to a broad user base.